Signal transduction is the process by which extracellular molecules influence intracellular events. A cellular response generally involves multiple signal transduction cascades that operate in concert to elicit a specific biological response. Activities of signal transduction pathways can be regulated through the use of biological molecules, i.e. protein kinases. While many of the proteins in these pathways are known, little is understood about the relationship between particular signal transduction pathways.
In U.S. Pat. No. 5,830,462, issued Nov. 3, 1998; and U.S. Pat. No. 5,834,266, issued Nov. 10, 1998, both to Crabtree et al (both incorporated herein by reference hereto), a general procedure for the regulation of dimerization and oligomerization of intracellular proteins as biological, control mechanisms is suggested. Crabtree further teaches that many signaling pathways originate with the binding of extracellular ligands to cell surface receptors, and receptor dimerization can lead to transphosphorylation and the recruitment of proteins that continue the signal transduction cascade.
In “Activation of the Raf-1 Kinase Cascade by Coumermycin Induced Dimerization,” Farrar, M. A., Alberola-Ila, J. and Perlmutter, R. M., Nature, Vol. 383, pp. 178-181, Sep. 12, 1996 (incorporated herein by reference hereto), the carboxy terminus of Raf-1 serine/threonine kinase was covalently attached to the amino terminus of the B subunit of bacterial DNA gyrase (GyrB). Streptomyces derived natural products, coumermycin and novobiocin, are known to bind GyrB with strong affinity. The region that binds the protein is known to be an oligosaccharide (also known as noviose sugar) and the attached coumarin, a common element between the 2 structures. The stoichiometry of binding between GyrB and drug is 2:1 for coumermycin and 1:1 for novobiocin.
Farrar et al suggests adding coumermycin to the chimeric Raf-GyrB fusion protein wherein, coumermycin bound 2 molecules of GyrB that concomitantly brought the 2 Raf kinases into close proximity to one another. This close proximity induced Raf dimerization and led to activation of the Raf kinase cascade.
Furthermore, Farrar et al teaches that if a sufficient concentration of novobiocin is added to the coumermycin/GyrB/Raf complex, coumermycin can be displaced from the GyrB active site having similar binding constants and block kinase activation.
Coumermycin is known to have induced dimerization of the tyrosine kinase, Jak2, as well as the transcription factors STAT3, STAT5a, and STAT5b. Unfortunately, it has failed to activate a number of other proteins involved in signal transduction. It is believed that GyrB-Coumermycin-GyrB complex can not orient all kinases and transcript factors into the proper orientation for cross-phosphorylation. A coumermycin analog with a more flexible or longer spacing group will result in the activation of additional signaling proteins, and might have enhanced cellular penetration and potency than coumermycin.